Cyclodextrin for the treatment of lysosomal storage diseases

ABSTRACT

The invention provides for methods of treating lysosomal storage disorders and/or reduction of non-cholesterol lipids, using cyclodextrin compounds, including in combination with other therapeutics, including vitamin E.

RELATED APPLICATIONS

This application claims priority of U.S. Provisional Application No.61/679,668, filed on Aug. 3, 2012, the entirety of which is incorporatedby reference herein.

FEDERALLY SPONSORED RESEARCH

This work was supported by the Federal Government. The Government hascertain rights in this invention.

TECHNICAL FIELD

The invention provides for methods of treating lysosomal storagedisorders and/or reduction of non-cholesterol lipids, using cyclodextrincompounds and cyclodextrin compounds in combination with other agents.

BACKGROUND OF THE INVENTION

Cyclodextrins (CD) are sugar molecules in a ring form. The alpha-CD (6sugars), beta-CD (7 sugars) and gamma-CD (8 sugars) are commonly usedcyclodextrins. The hydroxypropyl-beta cyclodextrin (HPβCD) has beenapproved for the pharmaceutical use as the drug excipient. Recentreports showed that beta-cyclodextrin including HPβCD andbeta-methyl-cyclodextrin (MβCD) reduced cholesterol accumulation andneuronal cell loss in the mouse model of Niemann Pick Type C (NPC)disease. The life span of these NPC KO mice also increased 80 to 100%after the CD treatment. The similar positive results were obtained inthe feline model of NPC disease. It was also reported that beta-CDincreased exocytosis in primary NPC fibroblasts.

It has been recently found that delta-tocopherol increased thecholesterol efflux from NPC cells and reduced cholesterol accumulation.Enhancement of lysosomal exocytosis has been indicated as a therapeuticstrategy for development of new treatment for all lysosomal storagediseases that are composed of 50 different diseases caused by thegenetic mutations of genes for lysosomal proteins. The phenotypicchanges in these diseases are accumulation of lipids, glycoproteinand/or other macromolecules in lysosomes and enlarged lysosome size inpatient cells that may lead to cell malfunction and cell death inaffected tissues.

SUMMARY OF THE INVENTION

In one aspect, the invention provides a method of treating a lysosomalstorage disorder in a subject, comprising determining that the subjectis in need of non-cholesterol lipid reduction or reduction ofnon-cholesterol dominant lipid and macromolecule accumulation, andadministering to the subject in need thereof, an effective amount of acyclodextrin compound, or a pharmaceutically acceptable salt, ester,solvate or hydrate thereof.

In another aspect, the invention provides a method of treating alysosomal storage disorder in a subject, wherein the subject has beenpreviously identified as being in need of non-cholesterol lipidreduction or reduction of non-cholesterol dominant lipid andmacromolecule accumulation, comprising administering to said subject inneed thereof an effective amount of a cyclodextrin compound, or apharmaceutically acceptable salt, ester, solvate or hydrate thereof.

In another aspect, the invention provides a method of treating alysosomal storage disorder in a subject, comprising the step ofadministering to the subject an effective amount of a cyclodextrincompound, or a pharmaceutically acceptable salt, ester, solvate orhydrate thereof, and an additional therapeutic agent.

In certain aspects, the invention provides a method of reducingnon-cholesterol lipids or reduction of non-cholesterol dominant lipidand macromolecule accumulation in a subject, the method comprisingadministering to the subject a cyclodextrin compound, or apharmaceutically acceptable salt, ester, solvate or hydrate thereof; anddetecting the amount of lipid reduction.

In another aspect, the invention provides a pharmaceutical compositioncomprising a cyclodextrin compound, or a pharmaceutically acceptablesalt, ester, solvate or hydrate thereof, and vitamin E, together with apharmaceutically-acceptable carrier or excipient.

In another aspect, the invention provides a method of treating a subjectsuffering from a lysosomal storage disorder, comprising the use of thepharmaceutical composition as described above, in combination withanother agent.

In one embodiment of any of the above aspects, the step of administeringthe cyclodextrin compound comprises administering the cyclodextrincompound to a subject such as a human in a dosage of between about 0.01mg/Kg/day and 100 mg/Kg/day.

In another embodiment of any of the above aspects, the cyclodextrincompound is administered to a subject such as a human in an amount fromabout 0.5 mg/Kg to 8 mg/Kg, either in a single dose or per day.

In one embodiment of any of the above aspects, the cyclodextrin compoundis administered to a subject such as a human in an amount of about 3mg/Kg either in a single dose or per day. In a further embodiment of anyone of the above aspects, the cyclodextrin compound is administered to asubject such as a human in an amount of about 1.0 mg/Kg, 1.25 mg/Kg, 1.5mg/Kg, 1.75 mg/Kg, 2.0 mg/Kg, 2.25 mg/Kg, 2.5 mg/Kg, 2.75 mg/Kg, 3.25mg/Kg, 3.5 mg/Kg, 3.75 mg/Kg, 4.0 mg/Kg, 4.25 mg/Kg, or 4.5 mg/Kg,either in a single dose or per day.

In another further embodiment of any of the above aspects, thecyclodextrin compound is administered to a subject such as a human in anamount from about 0.1 mg/Kg to 0.3 mg/Kg, 0.1 mg/Kg to 0.4 mg/Kg, 0.1mg/Kg to 0.5 mg/Kg, 0.1 mg/Kg to 0.6 mg/Kg, or 0.1 mg/Kg to 0.7 mg/Kg,either in a single dose or per day.

In another further embodiment of any of the above aspects, thecyclodextrin compound is administered in a single dose.

In one embodiment of any of the above aspects, the additionaltherapeutic agent (distinct from the cyclodextrin compound) isadministered to a subject such as a human in an amount from about 0.05to 1 mg/kg, either in a single dose or per day. In another embodiment ofany of the above aspects, the additional therapeutic agent (distinctfrom the cyclodextrin compound) is administered to a subject such as ahuman in an amount from about 0.1 mg/Kg to 0.3 mg/Kg, 0.1 mg/Kg to 0.4mg/Kg, 0.1 mg/Kg to 0.5 mg/Kg, 0.1 mg/Kg to 0.6 mg/Kg, or 0.1 mg/Kg to0.7 mg/Kg, either in a single dose or per day.

In one embodiment of any of the above aspects, the additionaltherapeutic agent is administered in a single dose.

In another aspect, the invention features a method of treating alysosomal storage disorder in a subject, comprising determining that thesubject is in need of non-cholesterol lipid reduction or reduction ofnon-cholesterol dominant lipid and macromolecule accumulation;administering to the subject in need thereof, an effective amount of acyclodextrin compound, or a pharmaceutically acceptable salt, ester,solvate or hydrate thereof in an amount from about 0.05 mg/Kg to 1 mg/Kgeither in a single dose or per day; and administering to the subject anadditional therapeutic agent (such as vitamin E) distinct from thecyclodextrin compound in an amount from about 0.05 mg/Kg to 1 mg/Kgeither in a single dose or per day.

In another aspect, the invention features a method of treating alysosomal storage disorder in a subject, comprising determining that thesubject is in need of non-cholesterol lipid reduction or reduction ofnon-cholesterol dominant lipid and macromolecule accumulation;administering to the subject in need thereof, an effective amount of acyclodextrin compound, or a pharmaceutically acceptable salt, ester,solvate or hydrate thereof; and administering to the subject anadditional therapeutic agent (such as vitamin E) distinct from thecyclodextrin compound.

In one embodiment, the cyclodextrin compound is administered to asubject such as a human in an amount from about 0.1 mg/Kg to 0.3 mg/Kg,0.1 mg/Kg to 0.4 mg/Kg, 0.1 mg/Kg to 0.5 mg/Kg, 0.1 mg/Kg to 0.6 mg/Kgor 0.1 mg/Kg to 0.7 mg/Kg, either in a single dose or per day.

In another embodiment, the additional therapeutic agent (distinct fromthe cyclodextrin compound) is administered to a subject such as a humanin an amount from about 0.1 mg/Kg to 0.3 mg/Kg, 0.1 mg/Kg to 0.4 mg/Kg,0.1 mg/Kg to 0.5 mg/Kg, 0.1 mg/Kg to 0.6 mg/Kg or 0.1 mg/Kg to 0.7mg/Kg, either in a single dose or per day.

In still another further embodiment, the cyclodextrin compound isadministered to a subject such as a human in an amount of about 50 uM incombination with 10 uM of an additional agent, either in a single doseor per day.

In a further preferred embodiment, the cyclodextrin compound isadministered to a subject such as a human in an amount of about 50 uM incombination with 10 uM of delta-tocopherol, either in a single dose orper day.

In other aspects, the invention provides a kit comprising an effectiveamount of a cyclodextrin compound, or a pharmaceutically acceptablesalt, ester, solvate or hydrate thereof, in unit dosage form, togetherwith instructions for administering the compound to a subject sufferingfrom a lysosomal storage disorder.

Various advantages of the invention include the following: Treatment ofall lysosomal storage diseases with cyclodextrins, includinghydroxypropyl-beta-cyclodextrin, but in certain aspects with theexception of Niemann Pick Type C disease; treatment of all lysosomalstorage diseases with cyclodextrins, includinghydroxypropyl-beta-cyclodextrin in combination of vitamin-E, forsynergistic or additive therapeutic effect, for reduction of dosage ofcyclodextrin needed that makes the cyclodextrin treatment morepractically feasible, and for less side effects by reducing dosages ofboth drugs; treatment of all lysosomal storage diseases withcyclodextrins (such as beta and gama forms) in combination with modifiedcyclodextrins for better efficacy and less side effects; treatment ofall lysosomal storage diseases with cyclodextrins and modified vitamin-Eanalogs for the better efficacy and less side effects.

Further, the invention provides the administration of the compounds ofthe invention for the treatment of all 40-50 lysosomal storage diseasesbased on the mechanism of action of cyclodextrin (increases lysosomalexocytosis).

Other aspects of the invention are disclosed infra.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a set of graphs showing the reduction of total cholesterol(including cholesterol ester and free cholesterol) in Wolman fibroblaststreated with δ-tocopherol, α-tocopherol, methyl-β-cyclodextrin, andcombinations of δ-tocopherol and methyl-β-cyclodextrin, and α-tocopheroland methyl-β-cyclodextrin (MBCD).

FIG. 2 is a set of photographs showing Nile Red staining of Wolmanfibroblasts treated with δ-tocopherol (D-T), α-tocopherol (a-T),methyl-β-cyclodextrin, and combinations of δ-tocopherol (D-T) andmethyl-β-cyclodextrin (MBCD), and α-tocopherol andmethyl-β-cyclodextrin.

FIG. 3 is a set of graphs showing exocytosis levels in Wolmanfibroblasts as measured by HEXB secretion.

FIG. 4 is a set of graphs showing lysosomal calcium efflux in wild-typefibroblasts and lysosomal storage disease fibroblasts in the presenceand absence of δ-tocopherol, α-tocopherol, methyl-β-cyclodextrin, andcombinations of δ-tocopherol and methyl-β-cyclodextrin, and α-tocopheroland methyl-β-cyclodextrin.

FIGS. 5A-5D are sets of photographs showing the effect of various formsof Cyclodextrins (a-CD, r-CD), δ-Tocopherol (D-T), and combinations inseven disease and wild-type fibroblast cell lines as measured using theLysotracker assay. Highly branched cyclodextrin (HBCD), also known asKleptose.

FIG. 6 is a set of photographs showing that cyclodextrin alleviatespathological ultrastructural changes in Wolman disease cells. methyl-βcyclodextrin (MBCD), δ-tocopherol (δ-toco).

FIG. 7 is a set of photographs showing the electron microscopic analysisof Farber fibroblasts treated with methyl-β cyclodextrin (MBCD),α-cyclodextrin (alpha CD), or γ-cyclodextrin (gamma-CD).

FIG. 8 is a set of photographs showing the electron microscopic analysisof Tay-Sach, Fabry, and Farber fibroblasts treated with methyl-βcyclodextrin (MBCD).

FIG. 9 is a set of photographs showing the electron microscopic analysisof Wolman, NPA, Batten, and MSIIIB fibroblasts treated with methyl-βcyclodextrin (MBCD).

FIG. 10 is a set of photographs showing the electron microscopicanalysis of Farber fibroblasts treated with δ-Tocopherol (DT) andmethyl-β cyclodextrin (MBCD); δ-Tocopherol (DT) and α-cyclodextrin(a-T); δ-Tocopherol (DT) and γ-cyclodextrin (gamma-CD); δ-Tocopherol(DT) and Kleptose (also known as HBCD); α-Tocopherol (a-T) and methyl-βcyclodextrin (MBCD); and α-Tocopherol (a-T) and Kleptose. KLEPTOSE orTRAPPSOL are the brand names of the chemical HBCD or HBPCD.

FIGS. 11 (A and B) is a set of photographs that shows the effects ofcyclodextrins and delta-tocopherol on reduction of cholesterolaccumulation (Amplex-re cholesterol assay and filipin staining) andenlarged lysosomes (Lysotracker staining) in the NPC1 skin fibroblasts.Methyl-β cyclodextrin (MBCD), HBPCD (Kleptose).

FIGS. 12 (A and B) shows the effects of cyclodextrins anddelta-tocopherol on reduction of cholesterol accumulation (Amplex-recholesterol assay and filipin staining) and enlarged lysosomes(Lysotracker staining) in the NPC1 neuronal cells (NPC1-NSCs). (A) showsconcentration-responses determined in Amplex-red cholesterol assay. (B)shows filipin and lysotracker staining. Methyl-β cyclodextrin (MBCD),δ-Tocopherol (δ-T)

FIGS. 13 (A and B) is a set of photographs that shows a comparison ofthe effect of single use of cyclodextrins with that in a combinationwith delta-tocopherol on reduction of cholesterol accumulation (filipinstaining) and enlarged lysosomes ((Lysotracker staining) in NPC1neuronal cells (NPC1-NSCs). (A) HBPCD+δ-Tocopherol. (B)MBCD+δ-Tocopherol.

FIG. 14 is a set of photographs showing lysotracker staining in 3123treated with methyl-β cyclodextrin (MBCD); HBPCD or δ-Tocopherol (DT).

FIG. 15 is a set of photographs showing lysotracker staining in ML111treated with methyl-β cyclodextrin (MBCD); HBPCD or δ-Tocopherol (DT).

FIG. 16 is a set of photographs showing lysotracker staining in MLIVtreated with methyl-β cyclodextrin (MBCD); HBPCD or δ-Tocopherol (DT).

FIG. 17 is a set of photographs showing lysotracker staining in MPS1treated with methyl-β cyclodextrin (MBCD); HBPCD or δ-Tocopherol (DT).

FIG. 18 is a set of photographs showing lysotracker staining in MPSV1treated with methyl-β cyclodextrin (MBCD); HBPCD or δ-Tocopherol (DT).

DETAILED DESCRIPTION

Methods of Treatment

In one aspect, the invention provides a method of treating a lysosomalstorage disorder in a subject, comprising determining that the subjectis in need of non-cholesterol lipid reduction or reduction ofnon-cholesterol dominant lipid and other macromolecule accumulation, andadministering to the subject in need thereof, an effective amount of acyclodextrin compound, or a pharmaceutically acceptable salt, ester,solvate or hydrate thereof.

In another aspect, the invention provides a method of treating alysosomal storage disorder in a subject, wherein the subject has beenpreviously identified as being in need of non-cholesterol lipidreduction or reduction of non-cholesterol dominant lipid and othermacromolecule accumulation, comprising administering to said subject inneed thereof an effective amount of a cyclodextrin compound, or apharmaceutically acceptable salt, ester, solvate or hydrate thereof.

In one embodiment, the lysosomal storage disorder is treated by reducingthe non-cholesterol lipid or non-cholesterol dominant lipid and othermacromolecule accumulation in the subject.

In another embodiment, the non-cholesterol lipid is lipopigments,globotriaosylceramide, ceramide, sphingomyelin, heparan sulfate,partially degraded heparan sulfate, GM2 ganglioside, triglycerides, orcholesterol esters. The other macromolecules include proteins,glycoproteins (sugar containing proteins), mucopolysaccharides (longunbranched polysaccharides), and other cellular components.

A subject may be identified as having a lysosomal storage disease bypresenting to a clinician with symptoms of a lysosomal storage disease,including but not limited to an enlarged liver and spleen. Storage maybegin during early embryonic development, and the clinical presentationfor lysosomal storage diseases can vary from an early and severephenotype to late-onset mild disease. Said subject may be subject to avariety of diagnostic tests to determine if the subject has a lysosomalstorage diease, and further to determine the presence of non-cholesterollipids and macromolecules and non-cholesterol dominant lipids (i.e.non-cholesterol lipids that are present in an amount or percentagegreater than that of cholesterol).

For example, ultrastructural examinations of skin biopsy specimens canbe used to detect lysosomal accumulation of undegraded metabolites. Atest of specific lysosomal enzyme activity can also be used to determinethe presence of specific lysosomal enzymes. Moreover, correlation ofboth skin ultrastructure and assay for specific lysosomal enzymes incultured dermal fibroblasts derived from the skin biopsy may alsofacilitate determination of cholesterol and non-cholesterol lipids, anddiagnostic accuracy. Filipin staining is a well-known histochemicalstain for cholesterol. Filipin is highly fluorescent and bindsspecifically to cholesterol. This method of detecting cholesterol incell membranes is used clinically, for example in the study anddiagnosis of Type C Niemann-Pick disease. Molecular genetic testing canbe used, may be use to refine the enzymatic diagnosis. Other diagnosticmethods to determine the present of cholesterol and non-cholesterollipids include antibody immunostaining or mass spectrometry.

Lysosomal storage disorders include ˜40 to 50 inherited metabolicdisorders caused by defects in lysosomal function. The incidence isabout 1:5000-1:10,000 as a group of diseases. The term lysosomal refersto a recycling center in which cell membrane and other materials breakdown to small molecules for reuse. It has been found that deficiency ofa single enzyme or proteins required for the metabolism or traffickingof lipids, glycoproteins and other macromolecules results in lipidaccumulation in lysosome of cells. Excessive amount of lipids or othermaterials in lysosome causes enlargement of liver and spleen. Symptomsof neuronal degeneration are common clinical manifestations in patientswith neuronal involvements.

Lysosomal storage disorders treated by the invention include, but arenot limited to the following: Aspartylglucosaminuria, Wolman disease,Cystinosis, Danon disease, Fabry disease, Farber disease, Fucosidosis,Gaucher disease, GM1-Gangliosidosis types I/II/III, GM2-Gangliosidosis,alpha-Mannosidosis types I/II, beta-Mannosidosis, Metachromaticleukodystrophy, Sialidosis types I/II, Mucolipidosis type IV, Scheiesyndrome, Hunter syndrome, Sanfilippo syndrome A, Sanfilippo syndrome B,Sanfilippo syndrome C, Sanfilippo syndrome D, Galactosialidosis typesI/II, Krabbe disease, Sandhoff disease, Vogt-Spielmeyer disease, Hurlersyndrome, Niemann-Pick disease, I-cell disease (mucolipidosis II),pseudo-Hurler polydystrophy, Morquio syndrome, Maroteaux-Lamy syndrome,Sly syndrome, Mucopolysaccharidosis type IX, Multiple sulfatasedeficiency, Batten disease, Tay-Sachs disease, Pompe disease, Battendisease, Batten disease, late infantile, Northern Epilepsy,Pycnodysostosis, Schindler disease, Sialuria, and Salla disease.

In certain embodiments, the lysosomal storage disorder is Tay-Sachsdisease, Sphingolipidoses, Gaucher disease, Mucolipidosis,Galactosialidosis, Salla disorder, Cystinosis, Danon disease, Fabrydisease, Farber disease, Lipofuscinoses, Pompe disease, Gangliodisosis,ISSD, Krabbe disease, leukodystrophy, Hurler disease, Scheie disease,Hunter disease, San Filippo disease, Sandhoff disease, Schinder disease,Batten disorder, or Wolman disease.

In a further embodiment, the lysosomal storage disorder is Tay-Sachsdisease, Fabry disease, Farber disease, San Filippo disease, Battendisorder, or Wolman disease.

In certain embodiments, the cyclodextrin compound is of formula (I):

or a pharmaceutically acceptable salt, ester, solvate or hydratethereof, wherein,

each R is independently H, alkyl, alkenyl, alkynyl, cycloalkyl,heterocycloalkyl, aryl, or heteroaryl, each of which is optionallysubstituted; or —C(O)OR^(B), —OC(O)R^(B), —C(O)R^(B), or—C(O)NR^(A)R^(B);

each R₁ is independently H, alkyl, alkenyl, alkynyl, cycloalkyl, aryl,heteroaryl, halogen, hydroxy, amino, —CN, —CF₃, —N₃, —NO₂, —OR^(B),—SR^(B), —SOR^(B), —SO₂R^(B), —N(R^(B))S(O₂)—R^(B), —N(R^(B))S(O₂)NR^(A)R^(B), —NR^(A)R^(B), —C(O)OR^(B), —OC(O)R^(B), —C(O)R^(B),—C(O)NR^(A)R^(B), or —N(R^(B))C(O)R^(B); each of which is optionallysubstituted;

each R^(A) is independently hydrogen, alkyl, alkenyl, alkynyl,cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each of which isoptionally substituted;

each R^(B) is independently hydrogen, alkyl, alkenyl, alkynyl,cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each of which isoptionally substituted;

n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and each m is independently 0, 1,2, 3, 4, or 5.

In certain embodiments, each R is independently H, optionallysubstituted alkyl, —C(O)OR^(B), —OC(O)R^(B), —C(O)R^(B), or—C(O)NR^(A)R^(B).

In a further embodiment, each R is independently H, methyl, ethyl,propyl, butyl, pentyl, hexyl, heptyl, or octyl; wherein each is straightchain or branched.

In other embodiments, n is 1, 2, or 3.

In another embodiment, the cyclodextrin is2-hydroxypropyl-β-cyclodextrin (2HPβCD), hydroxypropyl-β-cyclodextrin(HPβCD), methyl-β-cyclodextrin (MβCD), α-cyclodextrin, β-cyclodextrin,or γ-cyclodextrin, or a pharmaceutically acceptable salt, ester, solvateor hydrate thereof.

In certain embodiments, the compound is:

TABLE 1 Types of beta-cyclodextrins # of Name sugars R # of R2 NameHeptakis(2,6-di-o- 7 a combination of R1 = —H in 14 MβCD methyl)-beta-position 3 and R2 = —Me in cyclodextrin positions 2 and 6(2-Hydroxypropyl)- 7 a random combination of 1 to 10 Kleptosebeta-cyclodextrin R1 = —H and R2 = —CH—CHOH—CH3 being 4 the mostabundant species (2-Hydroxypropyl)- 7 a random combination of 4-10Trappsol beta-cyclodextrin R1 = —H and R2 = —CH—CHOH—CH3 being 7 themost abundant species

In certain embodiments, the step of determining non-cholesterol lipidreduction or non-cholesterol dominant lipid and macromoleculeaccumulation in a subject comprises any one or more of the following.

Amplex-Red Cholesterol Assay—

Total cholesterol in patient cells was measured by the Amplex-RedCholesterol Assay Kit (Invitrogen). The unesterified cholesterol wasdetermined using the same kit without the enzyme acid lipase. Esterifiedcholesterol was determined as the difference between the total andunesterified cholesterol values. The cells were seeded to black, tissueculture-treated 96-well, 384-well or 1536-well plates at 4000, 1000, 300cells/well in 100, 20 or 5 μl medium by a Multidrop Combi dispenser(Thermo Scientific, Waltham, Mass.) and cultured for 24 hr. The assayplates were added with compound dilution in DMSO solution using aPintool station (Klaypsys, San Diego, Calif.) and cultured for 3 days.The cells were washed twice manually for 96-well or 384-well plates orusing a centrifugation method in which the inverted plates were placedon a stack of paper towel and centrifuged at 800 rpm for 1 min followedby addition of 7 μl/well PBS (added gently with a 45 degree angledliquid dispenser (Klaypsys). The cholesterol assay mixture from the kitwas added at 100, 20 or 2.5 μl/well for 96-well, 384-well or 1536-wellplates and incubated for 1 hr at 37° C. The resulted fluorescenceintensity was measured with excitation of 560 (±10) and emission of 590(±10) in a fluorescence plate reader (Tecan, Durham, N.C.).

Nile-Red Staining—

The cells were cultured and treated as described above in 96-wellplates. On the experimental day, cells were washed two times with PBSand live-stained with 1 uM Nile-red dye solution (prepared in cellculture medium) at 100 μl/well followed by an incubation at 37° C. for10 min. After washed twice with PBS, the cells were fixed in 3.2%paraformaldehyde in PBS at 100 μl/well for 1 hr at RT. The nuclearstaining was carried out by an addition of 100 μl/well 1 μg/ml Hoechst33342 (Invitrogen) in PBS and incubation at RT for 30 min. The plate waswashed twice with PBS and the images were measured in Incell2000 imagingplate reader with a FITC filter set (Ex=480±20 nm and Ex=525±36 nm) forthe neutral lipids (cholesteryl esters and triglycerides) and a DAPIfilter set for Hoechst nuclear staining.

LysoTracker Dye Staining—

The assay was optimized to visualize the enlarged lysosomes by applyingappropriate concentration of LysoTracker dye in which the control cellsexhibited minimal staining while the disease cells showed significantstaining. The cells were cultured and treated as described above in96-well plates. On the experimental day, cells were live-stained with100 μl/well 50 nM LysoTracker-Red DND-99 dye (Invitrogen, # L-7528) inmedium at 37° C. for 1 hr followed by plate washing twice with PBS. Theplate was then fixed in 100 μl/well 3.2% formaldehyde for 1 hr andwashed for two times with PBS. The nuclear staining were carried out byan addition of 100 μl/well 1 μg/ml Hoechst 33342 (Invitrogen) in PBS andincubation at RT for 30 min. After washing twice with PBS, the plateswere stored at 4° C. until imaging analysis. DAPI filter set and TRITCfilter set in the Incell2000 imaging plate reader were used to visualizeHoechst nuclear staining and LysoTracker staining, respectively.

Measurement of β-Hexosaminidase (HEXB) Release—

Fibroblasts were cultured in 24-well plates at 30,000 cells/well in 0.4ml medium for one day at 37° C. After being washed twice with the assaybuffer (DMEM with 2 mM D-mannose 6-phosphate sodium salt), the cellswith 0.4 ml/well the assay buffer were incubated at 37° C. with 0.2ml/well compound in assay buffer. At the 5, 10, 20, 30 and 40 min timepoints, 30 μl of assay buffer from each well in the 24-well plate werealiquoted into a 96-well black plate. The rest of assay buffer in the24-well plate was discarded followed by addition of 0.6 ml 1%Triton-X100 in dH₂O to lyse the cells. After incubation at 37° C. for 30min, 6 μl/well cell lysate were added to the 96-well plate with 24 μlassay buffer followed by 90 μl/well 2.25 mM HEXB substrate,4-Methylumbelliferyl N-acetyl-β-D-glucosaminide (Sigma-Aldrich, #M2133), in a 25 mM citric acid buffer at pH 4.5. The 96-well plate wasthen measured in the Tecan fluorescence plate reader (Ex=365±20 nm andEm=460±20 nm) after 1 hr incubation at 37° C. and addition of 100μl/well stop solution (1 M glycine and 1 M NaOH at pH 10.5).

Intracellular and Lysosomal Ca²⁺ Measurements—

Intracellular cytosolic Ca²⁺ concentration was measured fluorescentlyusing a Fluo-8 dye kit (ATT Bioquest, Sunnyvale, Calif.) as describedpreviously. Briefly, fibroblasts were cultured at 2500 cell/well in 20μl medium in black, clear bottom 384-well plates for 24 hr at 37° C. Thecalcium dye mixture was added at 20 μl/well and incubated at 37° C. for30 min following by at RT for 30 min. The plates were then placed into afluorescence kinetic plate reader (μCell, Hamamatsu, Hamamatsu City,Japan). The basal fluorescence intensity was recorded 10 times at 1 Hzfor 10 seconds and the compound was then added at 20 μl/well inside theinstrument followed by additional reading at 1 Hz for 5 min. The resultswere normalized to the average basal fluorescence intensity in ratio andthe peak response (Max.) was used for the result calculation. Thelysosomal Ca²⁺ induced by Gly-Phe β-naphthylamide (GPN) was measuredsimilarly as that for cytosolic Ca²⁺ except 200 nM GPN was added insteadof 8-T or α-T after the measurement of basal fluorescence intensity thatreleased lysosomal Ca²⁺.

Electron Microscopy—

Fibroblast cells were seeded in 6-well plates at 150,000 cells/well in 5ml medium and cultured for 1 day in the presence or absence ofcompounds. Cells were fixed in 2% glutaraldehyde, 0.1 M cacodylatebuffer, pH 7.2 for 1 h at room temperature and then stored at 4° C.until TEM analysis was performed. The cells were post fixed in 1% osmiumtetroxide in the same buffer for 1 hour and en bloc stained with 0.5%uranyl acetate in 0.1 M acetate buffer, pH 4.2. The cells were thendehydrated in graded ethanol solutions (35%, 50%, 70%, 95% and 100%) andinfiltrated overnight in epoxy resin (Poly/Bed 812, Polysciences). Afteradding fresh pure resin the cell plates were cured for 72 h in 55° C.After removing the polystyrene plates, suitable areas for thinsectioning were selected, cut out with a jewelry saw and glued ontoempty resin stubs. About 70 nm thin sections were cut on anultramicrotome (Leica EM UC6) and mounted on naked copper grids. Thethin sections were double stained (uranyl acetate and lead citrate),examined in a Hitachi H-7650 transmission electron microscope, andimages were taken using an AMT CCD camera.

In various embodiments, the invention provides a method as describedabove further comprising the step of administering an additionaltherapeutic agent.

In certain embodiments, the additional therapeutic agent is a vitamin.

In a further embodiment, the additional therapeutic agent is vitamin E.

In other embodiments, the invention provides a method as described abovewherein the step of administering the cyclodextrin comprisesadministering the compound orally, topically, parentally, intravenouslyor intramuscularly.

In certain embodiments, the invention provides a method comprising thestep of administering an effective amount of the compound and apharmaceutically suitable excipient.

In certain embodiments, the invention provides a method as describedabove wherein the subject is a human.

In various embodiments, the step of administering the cyclodextrincomprises administering the compound to a subject such as a human in adosage of between about 0.01 μg/kg/day and 100 mg/kg/day, either in asingle dose or per day.

In another aspect, the invention provides a method of treating alysosomal storage disorder in a subject, comprising the step ofadministering to the subject an effective amount of a cyclodextrincompound, or a pharmaceutically acceptable salt, ester, solvate orhydrate thereof, and an additional therapeutic agent.

In certain embodiments, the additional therapeutic agent is a vitamin.

In a further embodiment, the additional therapeutic agent is vitamin E.

In certain aspects, the invention provides a method of reducingnon-cholesterol lipids in a subject, the method comprising administeringto the subject a cyclodextrin compound, or a pharmaceutically acceptablesalt, ester, solvate or hydrate thereof; and detecting the amount oflipid reduction.

In one embodiment, the subject is identified as being in need of lipidreduction.

In another aspect, the invention provides a pharmaceutical compositioncomprising a therapeutically effective amount of a compound of theinvention (any of the formulae presented herein), or a pharmaceuticallyacceptable salt, solvate or hydrate thereof, in combination with apharmaceutically acceptable carrier or excipient.

In one embodiment, the pharmaceutical composition is in combination witha vitamin. In a further embodiment, the vitamin is vitamin E.

In another aspect, the invention provides a pharmaceutical compositioncomprising a cyclodextrin compound, or a pharmaceutically acceptablesalt, ester, solvate or hydrate thereof, and vitamin E, together with apharmaceutically-acceptable carrier or excipient.

In one embodiment, the cyclodextrin compound is of formula (I), or apharmaceutically acceptable salt, ester, solvate or hydrate thereof.

In another embodiment, the cyclodextrin compound is2-hydroxypropyl-β-cyclodextrin (2HPβCD), hydroxypropyl-β-cyclodextrin(HPβCD), methyl-β-cyclodextrin (MβCD), α-cyclodextrin, β-cyclodextrin,or γ-cyclodextrin, or a pharmaceutically acceptable salt, ester, solvateor hydrate thereof.

In another aspect, the invention provides a method of treating a subjectsuffering from a lysosomal storage disorder, comprising the use of thepharmaceutical composition as described above, in combination withanother agent.

In other aspects, the invention provides a kit comprising an effectiveamount of a cyclodextrin compound, or a pharmaceutically acceptablesalt, ester, solvate or hydrate thereof, in unit dosage form, togetherwith instructions for administering the compound to a subject sufferingfrom a lysosomal storage disorder.

In one embodiment, the cyclodextrin compound is of formula (I), or apharmaceutically acceptable salt, ester, solvate or hydrate thereof.

In another embodiment, the cyclodextrin compound is2-hydroxypropyl-β-cyclodextrin (2HPβCD), hydroxypropyl-β-cyclodextrin(HPβCD), methyl-β-cyclodextrin (MβCD), α-cyclodextrin, β-cyclodextrin,or γ-cyclodextrin, or a pharmaceutically acceptable salt, ester, solvateor hydrate thereof.

In another embodiment, the kit further comprises vitamin E.

In another embodiment, the invention provides a method as describedabove further comprising the step of synthesizing or obtaining thecyclodextrin compounds. Yet another embodiment of the present inventionis a process of making any of the compounds delineated herein employingany of the synthetic means delineated herein, or using methods known toone of ordinary skill in the art.

In certain embodiments, cyclodextrins including α-, β- andγ-cyclodextrins increased intracellular Ca2+ and lysosomal exocytosis inboth wild type and cells with LSDs (Wolman disease).

In various embodiments, cyclodextrins reduced enlarged lysosomes in sixcell lines with LSDs.

In another embodiment, cyclodextrins reduced ultrastructural pathologicchanges in cells with Wolman diseases and other cells.

In certain embodiments, cyclodextrins in combination with tocopherolsynergistically/additively reduced cholesterol accumulation in cells ofNPC and Wolman diseases.

An inhibitory amount or dose of the compounds of the present inventionmay range from about 0.1 mg/kg to about 500 mg/kg, alternatively fromabout 1 to about 50 mg/kg. Inhibitory amounts or doses will also varydepending on route of administration, as well as the possibility ofco-usage with other agents.

The term “inhibitory amount” of a compound of the present inventionmeans a sufficient amount to decrease the disorder in a biologicalsample or a subject. It is understood that when said inhibitory amountof a compound of the present invention is administered to a subject itwill be at a reasonable benefit/risk ratio applicable to any medicaltreatment as determined by a physician. The term “biological sample(s),”as used herein, means a substance of biological origin, which may beintended for administration to a subject. Examples of biological samplesinclude, but are not limited to, blood and components thereof such asplasma, platelets, subpopulations of blood cells and the like; organssuch as kidney, liver, heart, lung, and the like; sperm and ova; bonemarrow and components thereof; or stem cells.

As referred to herein, the phrase “in combination with”, or “orconjunction with” when referring to administration of a cyclodextrincompound and an additional therapeutic agent (distinct from thecyclodextrin compound) such as a vitamin E compound is intended to referto all forms of administration that provide the cyclodextrin andadditional therapeutic compounds together, e.g. where the two compoundsare administered concurrently (e.g. in a single unitary formulation) orsequentially in any order. For instance, in a suitable aspect, forsequential administration, the cyclodextrin compound and the additionaltherapeutic agent may be formulated separately and administered withinabout 0.25, 0.5, 1, 2, 5, 10, 15, 20, 30, 40, 50 or 60 minutes or moreof each other. For sequential administration, preferably thecyclodextrin compound and the additional therapeutic agent may beformulated separately and administered within about 30, 20, 10 or 5minutes or less of each other.

Upon improvement of a subject's condition, a maintenance dose of acompound, composition or combination of this invention may beadministered, if necessary. Subsequently, the dosage or frequency ofadministration, or both, may be reduced, as a function of the symptoms,to a level at which the improved condition is retained when the symptomshave been alleviated to the desired level, treatment should cease. Thesubject may, however, require intermittent treatment on a long-termbasis upon any recurrence of disease symptoms.

It will be understood, however, that the total daily usage of thecompounds and compositions of the present invention will be decided bythe attending physician within the scope of sound medical judgment. Thespecific inhibitory dose for any particular patient will depend upon avariety of factors including the disorder being treated and the severityof the disorder; the activity of the specific compound employed; thespecific composition employed; the age, body weight, general health, sexand diet of the patient; the time of administration, route ofadministration, and rate of excretion of the specific compound employed;the duration of the treatment; drugs used in combination or coincidentalwith the specific compound employed; and like factors well known in themedical arts.

The total daily inhibitory dose of the compounds of this inventionadministered to a subject in single or in divided doses can be inamounts, for example, from 0.01 to 50 mg/kg body weight or more usuallyfrom 0.1 to 25 mg/kg body weight. Single dose compositions may containsuch amounts or submultiples thereof to make up the daily dose. In oneembodiment, treatment regimens according to the present inventioncomprise administration to a patient in need of such treatment fromabout 10 mg to about 1000 mg of the compound(s) of this invention perday in single or multiple doses. In another embodiment, the treatmentregimen comprises administration to a patient in need of such treatmentfrom about 25 mg to about 6000 mg of a compound(s) of this invention perday in single or multiple doses. For instance a compound of the presentinvention can be administered to a patient twice a day with a totaldaily dose of 4000, 4200, 4400, 4600, 4800 or 5000 mg.

A preferred single use of cyclodextrin for mammals including humans isfrom 0.1 mg/Kg to 8 mg/Kg, more preferably 0.5 mg/kG or 1.0 mg/Kg to 2mg/Kg, 3 mg/Kg, 4 mg/Kg, 5 mg/Kg, 6 mg/Kg, 7 mg/Kg or 84 mg/Kg. Onespecific preferred single use of cyclodextrin for mammals includinghumans is 3 mg/Kg.

In a combination therapy of a cyclodextrin compound administeredtogether or otherwise in conjuction with a vitamin E compound such asdelta-tocopherol, a preferred single dose for a mammal including a humanmay be from 0.05 to 1 mg/kg for each of the cyclodextrin compound andvitamin E compound (such as delta-tocopherol), more preferably 0.1 mg/Kgto 0.5 mg/Kg, 0.6 mg/Kg or 0.7 mg/Kg for each of a cyclodextrin compoundand vitamin E compound such as delta-tocopherol, still more preferably0.1 mg/Kg to 0.3 mg/Kg or 0.4 mg/Kg for each of a cyclodextrin compoundand vitamin E compound such as delta-tocopherol.

In embodiments of the present invention, treatment of a lysosomalstorage disorder with a combination of a cyclodextrin compound andvitamin E compound (such as delta-tocopherol) can allow for use of alower dosage of the cyclodextrin compound to achieve a therapeuticeffect than when the cyclodextrin compound is used alone. In certainaspects, the amount of the cyclodextrin compound administered is atleast 5%, at least 10%, at least 15%, at least 20%, at least 25%, atleast 30%, at least 35%, at least 40%, at least 50%, at least 55%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, or at least 90%, less than an amount of the cyclodextrincompound necessary to achieve a therapeutic effect if administeredwithout the vitamin E compound.

Biological Data

FIG. 1. (amplex red) Skin fibroblasts derived from NPC1 patientsdemonstrate profound and reproducible cholesterol accumulation in lateendosomes and lysosomes and, therefore, provide a robust cellular modelof NPC1 disease. Using a phenotypic screen with a biochemical assay(Amplex Red) to measure unesterified cholesterol; Delta-Tocopherol(“δ-T”; or “Delta-T”) δ-T was identified as a lead compound thatdramatically reduces cellular cholesterol accumulation in aconcentration dependent manner. We further evaluated effect ofcyclodextrins alone and in combination with delta Tocopherol in otherlysosomal storage disorders and we found that alpha-CD, beta-CD, andgamma-CD can reduce cholesterol accumulation, and MBCD was most potent.

FIG. 2. (Nile Red) Cells were cultured in the presence of deltatocopherol, alpha tocopherol and MBCD plus in combination for 3 days andthen stained for neutral lipid with Nile red. Delta Tocopherol and MBCDtreatment reduces accumulation of neutral lipids and it is morepronounced when used in combination. Alpha Tocopherol was not as potent.

FIG. 3. (Hex assay) Delta-T stimulates lysosomal exocytosis in Wolmanfibroblasts. 2-hydroxypropyl-beta-cyclodextrin has been reported topromote a calcium-dependent lysosomal exocytosis, which offers apotential mechanism for its cholesterol-reducing effect in LSDfibroblast. We measured lysosomal exocytosis in delta-T-treated Wolmanfibroblasts by determining the activity of beta-hexosaminidase (HEXB), alysosomal enzyme, in the extracellular medium. We found that HEXBactivity increased in culture medium after 40 uM δ-Tocopherol treatmentfor 24 hours compared with the vehicle treated cells. These resultsdemonstrate that the pharmacological effect of delta-T may be mediatedby the increase of cytosolic Ca²⁺ and enhancement of lysosomalexocytosis.

FIG. 4. (calcium assay) Delta-T increases intracellular Ca²⁺concentration and ameliorates lysosomal calcium deficiency in NPC1cells—Increase in the concentration of intracellular Ca²⁺, an importantsecond messenger, triggers a variety of cellular responses includinglysosomal exocytosis. In NPC1 fibroblasts there is a dysregulation ofcalcium homeostasis, as evidenced by lysosomal Ca²⁺ deficiency.Treatments that compensate for loss of lysosomal Ca²⁺ (e.g., curcumin)have been reported to reduce cholesterol storage in NPC1 cells. Toexplore whether delta-T may similarly exert its effects through changesin intracellular Ca²⁺⁺, we measured cytosolic calcium levels in bothNPC1 and Wolman cells following the treatment with δ-T. We found thatδ-T stimulated a transient increase of cytosolic Ca²⁺ in both NPC1 andWolman fibroblasts, as well as in control fibroblasts. In addition, theintracellular Ca²⁺ response to delta-T was independent of extracellularCa²⁺ concentration, indicating that Ca²⁺ was released from intracellularstorage sites such as the ER in response to δ-T. We further studied theeffect of delta-T on lysosomal Ca²⁺ released by Gly-Phe β-naphthylamide(GPN) in NPC1 fibroblasts. Consistent with an earlier report, lysosomalCa²⁺ was reduced in NPC1 cells compared with that in control cells.Treatment of NPC1 fibroblasts with 40 uM delta-T for 24 hourssignificantly increased lysosomal Ca²⁺.

FIG. 5. Based on the data for both NPC1 and Wolman cells, wehypothesized that the pharmacological effect of delta-T on theintracellular Ca²⁺ and lysosomal exocytosis is a general mechanism forelimination of lysosomal storage. To test this hypothesis we measuredthe ability of delta-T to decrease acidic/lysosomal compartment size asdetermined by LysoTracker staining in fibroblasts derived from patientswith six other diseases. Lysosomal storage occurs in these fibroblastsconsists of ceroid/lipofuscin in Batten (CLN2), globotriaosylceramide inFabry, ceramide in Farber, sphingomyelin in NPA, partially degradedheparan sulfate in Sanfilippo type B, and GM2 ganglioside in Tay-Sachs(Table S1). Whereas untreated fibroblasts showed increased LysoTrackerstaining, indicating the enlarged lysosomes, treatment with 40 uMdelta-T significantly reduced the LysoTracker staining in all sixfibroblast cell lines studied. Thus, the amelioration of lysosomalpathology by delta-T, initially demonstrated in NPC1 and Wolman cells,can be generalized to other lysosomal storage diseases.

The mixed lipid storage phenotype results in a marked enlargement oflysosomes in the NPC1 and Wolman fibroblasts. Therefore, we nextdetermined whether the enlarged lysosomes in these cells could bereduced by the treatment with δ-T. LysoTracker, a probe which stains theintracellular acidic compartment, has been used to visualize theenlarged endolysosomal compartment in NPC1 cells. We found increasedLysoTracker staining in both NPC1 and Wolman fibroblasts, as expected.Treatment with either 40 uM delta-T or 300 uM MBCD significantly reducedLysoTracker staining in both types of fibroblasts.

FIG. 6. Both NPC1 and Wolman fibroblasts have a distinct ultrastructuralphenotype that is evident by electron microscopy. The reduction ofacidic cellular compartments by delta-T treatment is consistent withdecreased intracellular storage that was confirmed by alleviation of theultrastructural pathology.

The electron microscopic images exhibited enlarged lysosomes full oflamellated membranes and dense osmiophilic material in NPC1 cells andlipid droplet-like and cleft-like lysosomes in Wolman cells. Treatmentwith 40 uM delta-T significantly reduced the characteristic storagematerials in lysosomes of both cell types. Together, these findingsdemonstrate that the delta-T-mediated cholesterol reduction isassociated with alleviation of the disease phenotypes in NPC1 and Wolmancells.

We have found that alpha-CD, beta-CD, and gamma-CD can reducecholesterol accumulation in NPC cells. We also found that these CDsincreased intracellular Ca2+ and enhanced exocytosis. The ranking orderof cholesterol reduction effect is MBCD>alpha-CD>gamma-CD. In additionwe found that the CD treatment reduced the pathological changes in theultrastructure of NPC cells using the electron microscopy analysis. Wealso found that CDs reduced enlarged lysosomes in the primaryfibroblasts of Wolman disease another lysosomal storage disease thatexhibits cholesterol ester accumulation due to the malfunction of acidlipase in lysosome. The electron microscopy data indicated that the CDeffect is more significant than that of delta-tocopherol in the Wolmancells.

We also found the synergy between CD and delta-tocopherol on the NPCcells and other 6 lysosomal storage disease cells including Wolman,Niemann Pick Type A, Farber, Tay-Sachs, MSIIIB and CLN2 (Batten)diseases. The fluorescence tagged CD study indicated that CD enters celland comes out of cell quickly, indicating via exocytosis. We also havethe data demonstrate that alpha-CD, beta-CD and gamma-CD enhanceexocytosis.

DEFINITIONS

Listed below are definitions of various terms used to describe thisinvention. These definitions apply to the terms as they are usedthroughout this specification and claims, unless otherwise limited inspecific instances, either individually or as part of a larger group.The number of carbon atoms in a hydrocarbyl substituent can be indicatedby the prefix “C_(x)-C_(y),” where x is the minimum and y is the maximumnumber of carbon atoms in the substituent. Likewise, a C_(x) chain meansa hydrocarbyl chain containing x carbon atoms.

The prefix “halo” indicates that the substituent to which the prefix isattached is substituted with one or more independently selected halogenradicals. For example, “C₁-C₆haloalkyl” means a C₁-C₆alkyl substituentwherein at least one hydrogen radical is replaced with a halogenradical.

If a linking element in a depicted structure is “absent” or “a bond”,then the left element in the depicted structure is directly linked tothe right element in the depicted structure. For example, if a chemicalstructure is depicted as X-(L)_(n)-Y wherein L is absent or n is 0, thenthe chemical structure is X—Y.

The term “alkyl” as used herein, refers to a saturated, straight- orbranched-chain hydrocarbon radical. For example, “C₁-C₈ alkyl” containsfrom one to eight carbon atoms. Examples of alkyl radicals include, butare not limited to, methyl, ethyl, propyl, isopropyl, n-butyl,tert-butyl, neopentyl, n-hexyl, heptyl, octyl radicals and the like.

The term “alkenyl” as used herein, denotes a straight- or branched-chainhydrocarbon radical containing one or more double bonds. For example,“C₂-C₈ alkenyl” contains from two to eight carbon atoms. Alkenyl groupsinclude, but are not limited to, for example, ethenyl, propenyl,butenyl, 1-methyl-2-buten-1-yl, heptenyl, octenyl and the like.

The term “alkynyl” as used herein, denotes a straight- or branched-chainhydrocarbon radical containing one or more triple bonds. For example,“C₂-C₈ alkynyl” contains from from two to eight carbon atoms.Representative alkynyl groups include, but are not limited to, forexample, ethynyl, 1-propynyl, 1-butynyl, heptynyl, octynyl and the like.

The term “cycloalkyl” denotes a monovalent group derived from amonocyclic or polycyclic saturated carbocyclic ring compound. Examplesof cycloalkyl include, but not limited to, cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, bicyclo[2.2.1]heptyl, and bicyclo[2.2.2]octyland the like. The terms “carbocycle” or “carbocyclic” or “carbocyclyl”refer to a saturated (e.g., “cycloalkyl”), partially saturated (e.g.,“cycloalkenyl” or “cycloalkynyl”) or completely unsaturated (e.g.,“aryl”) ring system containing zero heteroatom ring atom. A carbocyclylmay be, without limitation, a single ring, or two or more fused rings,or bridged or spiro rings. A carbocyclyl may contain, for example from 3to 10 ring members (i.e., C₃-C₁₀carbocyclyl, such as C₃-C₁₀cycloalkyl).A substituted carbocyclyl may have either cis or trans geometry.Representative examples of carbocyclyl groups include, but are notlimited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,cycloheptyl, cyclooctyl, cyclopentenyl, cyclopentadienyl,cyclohexadienyl, adamantyl, decahydro-naphthalenyl, octahydro-indenyl,cyclohexenyl, phenyl, naphthyl, fluorenyl, indanyl,1,2,3,4-tetrahydro-naphthyl, indenyl, isoindenyl, bicyclodecanyl,anthracenyl, phenanthrene, benzonaphthenyl (also known as “phenalenyl”),decalinyl, and norpinanyl and the like. A carbocyclyl group can beattached to the parent molecular moiety through any substitutable carbonatom of the group.

The term “aryl” refers to an aromatic carbocyclyl containing from 6 to14 carbon ring atoms. Non-limiting examples of aryls include phenyl,naphthalenyl, anthracenyl, and indenyl and the like. An aryl group canbe connected to the parent molecular moiety through any substitutablecarbon atom of the group.

The term “heteroaryl” means an aromatic heterocyclyl typicallycontaining from 5 to 18 ring atoms, wherein at least one ring atom is aheteroatom. A heteroaryl may be a single ring, or two or more fusedrings. Non-limiting examples of five-membered heteroaryls includeimidazolyl; furanyl; thiophenyl (or thienyl or thiofuranyl); pyrazolyl;oxazolyl; isoxazolyl; thiazolyl; 1,2,3-, 1,2,4-, 1,2,5-, and1,3,4-oxadiazolyl; and isothiazolyl. Non-limiting examples ofsix-membered heteroaryls include pyridinyl; pyrazinyl; pyrimidinyl;pyridazinyl; and 1,3,5-, 1,2,4-, and 1,2,3-triazinyl. Non-limitingexamples of 6/5-membered fused ring heteroaryls includebenzothiofuranyl, isobenzothiofuranyl, benzisoxazolyl, benzoxazolyl,purinyl, and anthranilyl. Non-limiting examples of 6/6-membered fusedring heteroaryls include quinolinyl; isoquinolinyl; and benzoxazinyl(including cinnolinyl and quinazolinyl).

The term “heterocycloalkyl” refers to a non-aromatic 3-, 4-, 5-, 6- or7-membered ring or a bi- or tri-cyclic group fused system, where atleast one of the ring atoms is a heteroatom, and where (i) each5-membered ring has 0 to 1 double bonds and each 6-membered ring has 0to 2 double bonds, (ii) the nitrogen and sulfur heteroatoms mayoptionally be oxidized, (iii) the nitrogen heteroatom may optionally bequaternized, and (iv) any of the above rings may be fused to a benzenering. Representative heterocycloalkyl groups include, but are notlimited to, [1,3]dioxolane, pyrrolidinyl, pyrazolinyl, pyrazolidinyl,imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, oxazolidinyl,isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, andtetrahydrofuryl and the like.

The terms “heterocyclic” or “heterocycle” or “heterocyclyl” refer to asaturated (e.g., “heterocycloalkyl”), partially unsaturated (e.g.,“heterocycloalkenyl” or “heterocycloalkynyl”) or completely unsaturated(e.g., “heteroaryl”) ring system, where at least one of the ring atomsis a heteroatom (i.e., nitrogen, oxygen or sulfur), with the remainingring atoms being independently selected from the group consisting ofcarbon, nitrogen, oxygen and sulfur. A heterocyclyl group can be linkedto the parent molecular moiety via any substitutable carbon or nitrogenatom in the group, provided that a stable molecule results. Aheterocyclyl may be, without limitation, a single ring. Non-limitingexamples of single-ring heterocyclyls include furanyl, dihydrofuranyl,pyrrolyl, isopyrrolyl, pyrrolinyl, pyrrolidinyl, imidazolyl,isoimidazolyl, imidazolinyl, imidazolidinyl, pyrazolyl, pyrazolinyl,pyrazolidinyl, triazolyl, tetrazolyl, dithiolyl, oxathiolyl, oxazolyl,isoxazolyl, thiazolyl, isothiazolyl, thiazolinyl, isothiazolinyl,thiazolidinyl, isothiazolidinyl, thiodiazolyl, oxathiazolyl, oxadiazoly,pyranyl, dihydropyranyl, pyridinyl, piperidinyl, pyridazinyl,pyrimidinyl, pyrazinyl, piperazinyl, triazinyl, isoxazinyl,oxazolidinyl, isoxazolidinyl, oxathiazinyl, oxadiazinyl, morpholinyl,azepinyl, oxepinyl, thiepinyl, or diazepinyl. A heterocyclyl may alsoinclude, without limitation, two or more rings fused together, such as,for example, naphthyridinyl, thiazolpyrimidinyl, thienopyrimidinyl,pyrimidopyrimidinyl, or pyridopyrimidinyl. A heterocyclyl may compriseone or more sulfur atoms as ring members; and in some cases, the sulfuratom(s) is oxidized to SO or SO₂. The nitrogen heteroatom(s) in aheterocyclyl may or may not be quaternized, and may or may not beoxidized to N-oxide. In addition, the nitrogen heteroatom(s) may or maynot be N-protected.

The terms “optionally substituted”, “optionally substituted alkyl,”“optionally substituted “optionally substituted alkenyl,” “optionallysubstituted alkynyl”, “optionally substituted carbocyclic,” “optionallysubstituted aryl”, “optionally substituted heteroaryl,” “optionallysubstituted heterocyclic,” and any other optionally substituted group asused herein, refer to groups that are substituted or unsubstituted byindependent replacement of one, two, or three or more of the hydrogenatoms thereon with typical substituents including, but not limited to:

-alkyl, -alkenyl, -alkynyl, -aryl, -arylalkyl, -heteroaryl,-heteroarylalkyl, -heterocycloalkyl, -cycloalkyl, -carbocyclic,-heterocyclic,

—F, —Cl, —Br, —I,

—OH, protected hydroxy, alkoxy, oxo, thiooxo,

—NO₂, —CN, CF₃, N₃,

—NH₂, protected amino, —NH alkyl, —NH alkenyl, —NH alkynyl, —NHcycloalkyl, —NH— aryl, —NH— heteroaryl, —NH— heterocyclic,-dialkylamino, -diarylamino, -diheteroarylamino,

—O— alkyl, —O— alkenyl, —O— alkynyl, —O— cycloalkyl, —O-aryl,—O-heteroaryl, —O— heterocyclic,

—C(O)— alkyl, —C(O)— alkenyl, —C(O)— alkynyl, —C(O)— cycloalkyl,—C(O)-aryl, —C(O)— heteroaryl, —C(O)-heterocycloalkyl,

—CONH₂, —CONH— alkyl, —CONH— alkenyl, —CONH— alkynyl, —CONH— cycloalkyl,—CONH-aryl, —CONH-heteroaryl, —CONH-heterocycloalkyl,

—OCO₂— alkyl, —OCO₂— alkenyl, —OCO₂— alkynyl, —OCO₂— cycloalkyl,—OCO₂-aryl, —OCO₂-heteroaryl, —OCO₂-heterocycloalkyl, —OCONH₂, —OCONH—alkyl, —OCONH— alkenyl, —OCONH— alkynyl, —OCONH— cycloalkyl, —OCONH—aryl, —OCONH— heteroaryl, —OCONH— heterocycloalkyl,

—NHC(O)— alkyl, —NHC(O)— alkenyl, —NHC(O)— alkynyl, —NHC(O)— cycloalkyl,—NHC(O)-aryl, —NHC(O)-heteroaryl, —NHC(O)-heterocycloalkyl, —NHCO₂—alkyl, —NHCO₂-alkenyl, —NHCO₂— alkynyl, —NHCO₂-cycloalkyl, —NHCO₂— aryl,—NHCO₂— heteroaryl, —NHCO₂— heterocycloalkyl, —NHC(O)NH₂, —NHC(O)NH—alkyl, —NHC(O)NH— alkenyl, —NHC(O)NH— alkenyl, —NHC(O)NH— cycloalkyl,—NHC(O)NH-aryl, —NHC(O)NH-heteroaryl, —NHC(O)NH-heterocycloalkyl,NHC(S)NH₂, —NHC(S)NH— alkyl, —NHC(S)NH— alkenyl, —NHC(S)NH— alkynyl,—NHC(S)NH— cycloalkyl, —NHC(S)NH-aryl, —NHC(S)NH-heteroaryl,—NHC(S)NH-heterocycloalkyl, —NHC(NH)NH₂, —NHC(NH)NH— alkyl,—NHC(NH)NH-alkenyl, —NHC(NH)NH— alkenyl, —NHC(NH)NH— cycloalkyl,—NHC(NH)NH-aryl, —NHC(NH)NH-heteroaryl, —NHC(NH)NH-heterocycloalkyl,—NHC(NH)— alkyl, —NHC(NH)— alkenyl, —NHC(NH)— alkenyl, —NHC(NH)—cycloalkyl, —NHC(NH)-aryl, —NHC(NH)— heteroaryl,—NHC(NH)-heterocycloalkyl,

—C(NH)NH— alkyl, —C(NH)NH— alkenyl, —C(NH)NH— alkynyl, —C(NH)NH—cycloalkyl, —C(NH)NH-aryl, —C(NH)NH-heteroaryl,—C(NH)NH-heterocycloalkyl,

—S(O)— alkyl, —S(O)— alkenyl, —S(O)— alkynyl, —S(O)— cycloalkyl,—S(O)-aryl, —S(O)-heteroaryl, —S(O)-heterocycloalkyl —SO₂NH₂, —SO₂NH—alkyl, —SO₂NH— alkenyl, —SO₂NH-alkynyl, —SO₂NH— cycloalkyl, —SO₂NH—aryl, —SO₂NH— heteroaryl, —SO₂NH— heterocycloalkyl,

—NHSO₂— alkyl, —NHSO₂— alkenyl, —NHSO₂— alkynyl, —NHSO₂— cycloalkyl,—NHSO₂-aryl, —NHSO₂-heteroaryl, —NHSO₂-heterocycloalkyl,

—CH₂NH₂, —CH₂SO₂CH₃, polyalkoxyalkyl, polyalkoxy, -methoxymethoxy,-methoxyethoxy, —SH, —S— alkyl, —S— alkenyl, —S— alkynyl, —S—cycloalkyl, —S-aryl, —S— heteroaryl, —S-heterocycloalkyl, ormethylthiomethyl.

It is understood that the aryls, heteroaryls, carbocycles, heterocycles,alkyls, and the like can be further substituted.

The terms “halo” and “halogen,” as used herein, refer to an atomselected from fluorine, chlorine, bromine and iodine.

The term “subject” as used herein refers to a mammal. A subjecttherefore refers to, for example, dogs, cats, horses, cows, pigs, guineapigs, and the like. Preferably the subject is a human. When the subjectis a human, the subject may be either a patient or a healthy human.

The term “non-cholesterol lipid” is meant to refer to any lipid that isnot cholesterol, for example a macromolecule. Exemplary non-cholestorllipids include, but are not limited to, lipopigments,globotriaosylceramide, ceramide, sphingomyelin, heparan sulfate,partially degraded heparan sulfate, GM2 ganglioside, triglycerides, andcholesterol esters, and derivatives thereof. A “non-cholesterol dominantlipid” is meant to refer to any lipid that is not cholesterol, that ispresent in an amount greater than cholesterol making it thenon-cholesterol dominant lipid.

The term “leaving group,” or “LG”, as used herein, refers to any groupthat leaves in the course of a chemical reaction involving the group andincludes but is not limited to halogen, brosylate, mesylate, tosylate,triflate, p-nitrobenzoate, phosphonate groups, for example.

The term “protected hydroxy,” as used herein, refers to a hydroxy groupprotected with a hydroxy protecting group, as defined above, includingbenzoyl, acetyl, trimethylsilyl, triethylsilyl, methoxymethyl groups,for example.

The term “hydroxy protecting group,” as used herein, refers to a labilechemical moiety which is known in the art to protect a hydroxy groupagainst undesired reactions during synthetic procedures. After saidsynthetic procedure(s) the hydroxy protecting group as described hereinmay be selectively removed. Hydroxy protecting groups as known in theare described generally in T. H. Greene and P. G. M. Wuts, ProtectiveGroups in Organic Synthesis, 3rd edition, John Wiley & Sons, New York(1999). Examples of hydroxy protecting groups include benzyloxycarbonyl,4-nitrobenzyloxycarbonyl, 4-bromobenzyloxycarbonyl,4-methoxybenzyloxycarbonyl, methoxycarbonyl, tert-butoxycarbonyl,isopropoxycarbonyl, diphenylmethoxycarbonyl,2,2,2-trichloroethoxycarbonyl, 2-(trimethylsilyl)ethoxycarbonyl,2-furfuryloxycarbonyl, allyloxycarbonyl, acetyl, formyl, chloroacetyl,trifluoroacetyl, methoxyacetyl, phenoxyacetyl, benzoyl, methyl, t-butyl,2,2,2-trichloroethyl, 2-trimethylsilyl ethyl, 1,1-dimethyl-2-propenyl,3-methyl-3-butenyl, allyl, benzyl, para-methoxybenzyldiphenylmethyl,triphenylmethyl(trityl), tetrahydrofuryl, methoxymethyl,methylthiomethyl, benzyloxymethyl, 2,2,2-triehloroethoxymethyl,2-(trimethylsilyl)ethoxymethyl, methanesulfonyl, para-toluenesulfonyl,trimethylsilyl, triethylsilyl, triisopropylsilyl, and the like.Preferred hydroxy protecting groups for the present invention are acetyl(Ac or —C(O)CH₃), benzoyl (Bz or —C(O)C₆H₅), and trimethylsilyl (TMS or—Si(CH₃)₃).

The term “amino protecting group,” as used herein, refers to a labilechemical moiety which is known in the art to protect an amino groupagainst undesired reactions during synthetic procedures. After saidsynthetic procedure(s) the amino protecting group as described hereinmay be selectively removed Amino protecting groups as known in the aredescribed generally in T. H. Greene and P. G. M. Wuts, Protective Groupsin Organic Synthesis, 3rd edition, John Wiley & Sons, New York (1999).Examples of amino protecting groups include, but are not limited to,t-butoxycarbonyl, 9-fluorenylmethoxycarbonyl, benzyloxycarbonyl, and thelike.

The term “protected amino,” as used herein, refers to an amino groupprotected with an amino protecting group as defined above.

The term “alkylamino” refers to a group having the structure—N(R_(a)R_(b)), where R_(a) and R_(b) are independent H or alkyl.

As used herein, the term “pharmaceutically acceptable salt” refers tothose salts of the compounds formed by the process of the presentinvention which are, within the scope of sound medical judgment,suitable for use in contact with the tissues of humans and lower animalswithout undue toxicity, irritation, allergic response and the like, andare commensurate with a reasonable benefit/risk ratio. Pharmaceuticallyacceptable salts are well known in the art. For example, S. M. Berge, etal. describes pharmaceutically acceptable salts in detail in J.Pharmaceutical Sciences, 66: 1-19 (1977). The salts can be prepared insitu during the final isolation and purification of the compounds of theinvention, or separately by reacting the free base function with asuitable organic acid. Examples of pharmaceutically acceptable saltsinclude, but are not limited to, nontoxic acid addition salts, or saltsof an amino group formed with inorganic acids such as hydrochloric acid,hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid orwith organic acids such as acetic acid, maleic acid, tartaric acid,citric acid, succinic acid or malonic acid or by using other methodsused in the art such as ion exchange. Other pharmaceutically acceptablesalts include, but are not limited to, adipate, alginate, ascorbate,aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate,camphorate, camphorsulfonate, citrate, cyclopentanepropionate,digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate,glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate,hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate,lactate, laurate, lauryl sulfate, malate, maleate, malonate,methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate,oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate,phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate,tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts,and the like. Representative alkali or alkaline earth metal saltsinclude sodium, lithium, potassium, calcium, or magnesium salts, and thelike. Further pharmaceutically acceptable salts include, whenappropriate, nontoxic ammonium, quaternary ammonium, and amine cationsformed using counterions such as halide, hydroxide, carboxylate,sulfate, phosphate, nitrate, alkyl having from 1 to 6 carbon atoms,sulfonate and aryl sulfonate.

As used herein, the term “pharmaceutically acceptable ester” refers toesters of the compounds formed by the process of the present inventionwhich hydrolyze in vivo and include those that break down readily in thehuman body to leave the parent compound or a salt thereof. Suitableester groups include, for example, those derived from pharmaceuticallyacceptable aliphatic carboxylic acids, particularly alkanoic, alkenoic,cycloalkanoic and alkanedioic acids, in which each alkyl or alkenylmoiety advantageously has not more than 6 carbon atoms. Examples ofparticular esters include, but are not limited to, formates, acetates,propionates, butyrates, acrylates and ethylsuccinates.

The term “pharmaceutically acceptable prodrugs” as used herein refers tothose prodrugs of the compounds formed by the process of the presentinvention which are, within the scope of sound medical judgment,suitable for use in contact with the tissues of humans and lower animalswith undue toxicity, irritation, allergic response, and the like,commensurate with a reasonable benefit/risk ratio, and effective fortheir intended use, as well as the zwitterionic forms, where possible,of the compounds of the present invention. “Prodrug”, as used hereinmeans a compound which is convertible in vivo by metabolic means (e.g.by hydrolysis) to afford any compound delineated by the formulae of theinstant invention. Various forms of prodrugs are known in the art, forexample, as discussed in Bundgaard, (ed.), Design of Prodrugs, Elsevier(1985); Widder, et al. (ed.), Methods in Enzymology, vol. 4, AcademicPress (1985); Krogsgaard-Larsen, et al., (ed). “Design and Applicationof Prodrugs, Textbook of Drug Design and Development, Chapter 5, 113-191(1991); Bundgaard, et al., Journal of Drug Deliver Reviews,8:1-38(1992); Bundgaard, J. of Pharmaceutical Sciences, 77:285 et seq.(1988); Higuchi and Stella (eds.) Prodrugs as Novel Drug DeliverySystems, American Chemical Society (1975); and Bernard Testa & JoachimMayer, “Hydrolysis In Drug And Prodrug Metabolism: Chemistry,Biochemistry And Enzymology,” John Wiley and Sons, Ltd. (2002).

This invention also encompasses pharmaceutical compositions containingpharmaceutically acceptable prodrugs of compounds of the invention. Forexample, compounds of the invention having free amino, amido, hydroxy orcarboxylic groups can be converted into prodrugs. Prodrugs includecompounds wherein an amino acid residue, or a polypeptide chain of twoor more (e.g., two, three or four) amino acid residues is covalentlyjoined through an amide or ester bond to a free amino, hydroxy orcarboxylic acid group of compounds of the invention. The amino acidresidues include but are not limited to the 20 naturally occurring aminoacids commonly designated by three letter symbols and also includes4-hydroxyproline, hydroxylysine, demosine, isodemosine,3-methylhistidine, norvalin, beta-alanine, gamma-aminobutyric acid,citrulline, homocysteine, homoserine, ornithine and methionine sulfone.Additional types of prodrugs are also encompassed. For instance, freecarboxyl groups can be derivatized as amides or alkyl esters. Freehydroxy groups may be derivatized using groups including but not limitedto hemisuccinates, phosphate esters, dimethylaminoacetates, andphosphoryloxymethyloxy carbonyls, as outlined in Advanced Drug DeliveryReviews, 1996, 19, 1 15. Carbamate prodrugs of hydroxy and amino groupsare also included, as are carbonate prodrugs, sulfonate esters andsulfate esters of hydroxy groups. Derivatization of hydroxy groups as(acyloxy)methyl and (acyloxy)ethyl ethers wherein the acyl group may bean alkyl ester, optionally substituted with groups including but notlimited to ether, amine and carboxylic acid functionalities, or wherethe acyl group is an amino acid ester as described above, are alsoencompassed. Prodrugs of this type are described in J. Med. Chem. 1996,39, 10. Free amines can also be derivatized as amides, sulfonamides orphosphonamides. All of these prodrug moieties may incorporate groupsincluding but not limited to ether, amine and carboxylic acidfunctionalities.

As used herein, “solvate” refers to the physical association of acompound of the invention with one or more solvent molecule, whetherorganic or inorganic. This physical association often includes hydrogenbonding. In certain instances, the solvate is capable of isolation, forexample, when one or more solvate molecules are incorporated in thecrystal lattice of the crystalline solid.

Combinations of substituents and variables envisioned by this inventionare only those that result in the formation of stable compounds. Theterm “stable”, as used herein, refers to compounds which possessstability sufficient to allow manufacture and which maintains theintegrity of the compound for a sufficient period of time to be usefulfor the purposes detailed herein (e.g., therapeutic or prophylacticadministration to a subject).

Pharmaceutical Compositions

The pharmaceutical compositions of the present invention comprise atherapeutically effective amount of a compound of the present inventionformulated together with one or more pharmaceutically acceptablecarriers. As used herein, the term “pharmaceutically acceptable carrier”means a non-toxic, inert solid, semi-solid or liquid filler, diluent,encapsulating material or formulation auxiliary of any type. Thepharmaceutical compositions of this invention can be administered tohumans and other animals orally, rectally, parenterally,intracisternally, intravaginally, intraperitoneally, topically (as bypowders, ointments, or drops), buccally, or as an oral or nasal spray.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, microemulsions, solutions, suspensions, syrups,and elixirs. In addition to the active compounds, the liquid dosageforms may contain inert diluents commonly used in the art such as, forexample, water, alcohol or other solvents, solubilizing agents andemulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate,ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol,1,3-butylene glycol, polysorbate, dimethylformamide, oils (inparticular, cottonseed, groundnut, corn, germ, olive, castor, and sesameoils), mono- or diglycerides, glycerol, tetrahydrofurfuryl alcohol,polyethylene glycols and fatty acid esters of sorbitan, and mixturesthereof. Besides inert diluents, the oral compositions can also includeadjuvants such as wetting agents, emulsifying and suspending agents,antioxidants, sweetening, flavoring, and perfuming agents. The liquiddosage form can also be encapsulated in a gelatin capsule, wherein acompound of the present invention can be dissolved in a pharmaceuticallyacceptable carrier containing, for example, one or more solubilizatingagents (e.g., polysorbate 80 and mono and diglycerides), and othersuitable excipients (e.g., an antioxidants such as ascorbyl palmitate,or a sweetening or flavoring agent).

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions may be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation may also be a sterile injectablesolution, suspension or emulsion in a nontoxic parenterally acceptablediluent or solvent, for example, as a solution in 1,3-butanediol. Amongthe acceptable vehicles and solvents that may be employed are water,Ringer's solution, U.S.P. and isotonic sodium chloride solution. Inaddition, sterile, fixed oils are conventionally employed as a solventor suspending medium. For this purpose any bland fixed oil can beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid are used in the preparation of injectables.

In certain preferred embodiments, the compositions of the presentinvention are administered intracranially, for instance injected intothe brain, such as by direct injection into the brain. Direct injectionmay be performed by intraventricular and intracerebral routes. Injectionof the compositions into the brain can also be performed using a devicefor administration. Because cyclodextrin and delta-tocopherol may pose achallenge with brain penetration and quick drug metabolism, directadministration of the drugs into the central nervous system may beachieved by using epidural (injection or infusion into the epiduralspace), intracerebral (into the cerebrum), intracerebroventricular (intothe cerebral ventricles), or intrathecal (into the spinal canal)injection. Pathan et al. (Recent Patents on Drug Delivery & Formulation2009, 3, 71-89), incorporated by reference in its entirety herein,describes some method of administration of a composition to the brain.

In order to prolong the effect of a drug, it is often desirable to slowthe absorption of the drug from subcutaneous or intramuscular injection.This may be accomplished by the use of a liquid suspension ofcrystalline or amorphous material with poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolutionwhich, in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally administered drugform is accomplished by dissolving or suspending the drug in an oilvehicle. Immediate release forms are also contemplated by the presentinvention.

Compositions for rectal or vaginal administration are preferablysuppositories which can be prepared by mixing the compounds of thisinvention with suitable non-irritating excipients or carriers such ascocoa butter, polyethylene glycol or a suppository wax which are solidat ambient temperature but liquid at body temperature and therefore meltin the rectum or vaginal cavity and release the active compound.

Solid compositions of a similar type may also be employed as fillers insoft and hard-filled gelatin capsules using such excipients as lactoseor milk sugar as well as high molecular weight polyethylene glycols andthe like.

The active compounds can also be in micro-encapsulated form with one ormore excipients as noted above.

The solid dosage forms of tablets, dragees, capsules, pills, andgranules can be prepared with coatings and shells such as entericcoatings, release controlling coatings and other coatings well known inthe pharmaceutical formulating art. In such solid dosage forms theactive compound may be admixed with at least one inert diluent such assucrose, lactose or starch. Such dosage forms may also comprise, as isnormal practice, additional substances other than inert diluents, e.g.,tableting lubricants and other tableting aids such a magnesium stearateand microcrystalline cellulose. In the case of capsules, tablets andpills, the dosage forms may also comprise buffering agents.

Preferably, a compound of the invention is formulated in a soliddispersion, where the compound can be molecularly dispersed in a matrixwhich comprises a pharmaceutically acceptable, hydrophilic polymer. Thematrix may also contain a pharmaceutically acceptable surfactant.Suitable solid dispersion technology for formulating a compound of theinvention includes, but is not limited to, melt extrusion, spray drying,or solvent evaporization.

Dosage forms for topical or transdermal administration of a compound ofthis invention include ointments, pastes, creams, lotions, gels,powders, solutions, sprays, inhalants or patches. The active componentis admixed under sterile conditions with a pharmaceutically acceptablecarrier and any needed preservatives or buffers as may be required.Ophthalmic formulation, ear drops, eye ointments, powders and solutionsare also contemplated as being within the scope of this invention.

The ointments, pastes, creams and gels may contain, in addition to anactive compound of this invention, excipients such as animal andvegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulosederivatives, polyethylene glycols, silicones, bentonites, silicic acid,talc and zinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to the compounds of thisinvention, excipients such as lactose, talc, silicic acid, aluminumhydroxide, calcium silicates and polyamide powder, or mixtures of thesesubstances. Sprays can additionally contain customary propellants suchas chlorofluorohydrocarbons.

Transdermal patches have the added advantage of providing controlleddelivery of a compound to the body. Such dosage forms can be made bydissolving or dispensing the compound in the proper medium. Absorptionenhancers can also be used to increase the flux of the compound acrossthe skin. The rate can be controlled by either providing a ratecontrolling membrane or by dispersing the compound in a polymer matrixor gel.

The compounds described herein contain one or more asymmetric centersand thus give rise to enantiomers, diastereomers, and otherstereoisomeric forms that may be defined, in terms of absolutestereochemistry, as (R)- or (S)-, or as (D)- or (L)- for amino acids.The present invention is meant to include all such possible isomers, aswell as their racemic and optically pure forms. Optical isomers may beprepared from their respective optically active precursors by theprocedures described above, or by resolving the racemic mixtures. Theresolution can be carried out in the presence of a resolving agent, bychromatography or by repeated crystallization or by some combination ofthese techniques which are known to those skilled in the art. Furtherdetails regarding resolutions can be found in Jacques, et al.,Enantiomers, Racemates, and Resolutions (John Wiley & Sons, 1981). Whenthe compounds described herein contain olefinic double bonds or othercenters of geometric asymmetry, and unless specified otherwise, it isintended that the compounds include both E and Z geometric isomers.Likewise, all tautomeric forms are also intended to be included. Theconfiguration of any carbon-carbon double bond appearing herein isselected for convenience only and is not intended to designate aparticular configuration unless the text so states; thus a carbon-carbondouble bond depicted arbitrarily herein as trans may be cis, trans, or amixture of the two in any proportion.

The synthesized compounds can be separated from a reaction mixture andfurther purified by a method such as column chromatography, highpressure liquid chromatography, or recrystallization. As can beappreciated by the skilled artisan, further methods of synthesizing thecompounds of the formulae herein will be evident to those of ordinaryskill in the art. Additionally, the various synthetic steps may beperformed in an alternate sequence or order to give the desiredcompounds. In addition, the solvents, temperatures, reaction durations,etc. delineated herein are for purposes of illustration only and one ofordinary skill in the art will recognize that variation of the reactionconditions can produce the desired bridged macrocyclic products of thepresent invention. Synthetic chemistry transformations and protectinggroup methodologies (protection and deprotection) useful in synthesizingthe compounds described herein are known in the art and include, forexample, those such as described in R. Larock, Comprehensive OrganicTransformations, VCH Publishers (1989); T. W. Greene and P. G. M. Wuts,Protective Groups in Organic Synthesis, 2d. Ed., John Wiley and Sons(1991); L. Fieser and M. Fieser, Fieser and Fieser's Reagents forOrganic Synthesis, John Wiley and Sons (1994); and L. Paquette, ed.,Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons(1995), and subsequent editions thereof.

The compounds of this invention may be modified by appending variousfunctionalities via any synthetic means delineated herein to enhanceselective biological properties. Such modifications are known in the artand include those which increase biological penetration into a givenbiological system (e.g., blood, lymphatic system, central nervoussystem), increase oral availability, increase solubility to allowadministration by injection, alter metabolism and alter rate ofexcretion.

The recitation of a listing of chemical groups in any definition of avariable herein includes definitions of that variable as any singlegroup or combination of listed groups. The recitation of an embodimentfor a variable herein includes that embodiment as any single embodimentor in combination with any other embodiments or portions thereof.

EXAMPLES

The compounds and processes of the present invention will be betterunderstood in connection with the following examples, which are intendedas an illustration only and not to limit the scope of the invention. Thefollowing examples can be prepared according to the schemes as describedabove, or according to the synthetic steps as described below. Variouschanges and modifications to the disclosed embodiments will be apparentto those skilled in the art and such changes and modificationsincluding, without limitation, those relating to the chemicalstructures, substituents, derivatives, formulations and/or methods ofthe invention may be made without departing from the spirit of theinvention and the scope of the appended claims.

The chemical structures herein contain certain —NH—, —NH₂ (amino) and—OH (hydroxyl) groups where the corresponding hydrogen atom(s) may notexplicitly appear; however they are to be read as —NH—, —NH₂ or —OH asthe case may be.

Example 1 δ-Tocopherol and Cyclodextrin Treatment Reduces LipidAccumulation in Lysosomes of Lysosome Storage Disorder Cells

The effect of methyl-β-cyclodextrin on lipid accumulation in fibroblastlines derived from patients with Wolman disease was investigated. Wolmanfibroblasts were treated with δ-tocopherol, α-tocopherol,methyl-β-cyclodextrin, or combinations of δ-tocopherol andmethyl-β-cyclodextrin or α-tocopherol and methyl-β-cyclodextrin. Totalcholesterol and free cholesterol were then measured using the Amplex-RedCholesterol Oxidase assay (Invitrogen) according to the manufacturer'sinstructions. As shown in FIGS. 1A-1C (Total cholesterol) and in FIGS.1D-1F (Free cholesterol), treatment with δ-tocopherol,methyl-β-cyclodextrin, or combinations of δ-tocopherol andmethyl-β-cyclodextrin or α-tocopherol and methyl-β-cyclodextrin caused asignificant reduction in total cholesterol and free cholesterol inWolman fibroblasts. To further characterize the effect of treatment onlipid accumulation, treated fibroblasts were evaluated using a Nile Redassay to measure neutral lipid accumulation. In brief, cells treatedwith various drugs were stained with Nile Red which selectively labelslipid accumulations within cells. The Nile Red staining was visualizedby fluorescent microscopy. As shown in FIG. 2, untreated Wolmanfibroblasts show cytoplasmic droplets of neutral lipid accumulation.These neutral lipid accumulations were significantly reduced upontreatment with δ-tocopherol or methyl-β-cyclodextrin. Interestingly,α-tocopherol treatment failed to show any effect on neutral lipidaccumulation.

The effects of δ-tocopherol, α-tocopherol, methyl-β-cyclodextrin, orcombinations of δ-tocopherol and methyl-β-cyclodextrin or α-tocopheroland methyl-β-cyclodextrin treatment on lysosomal exocytosis weredetermined using the HEXB assay. In brief, the level of lysosomalexocytosis was determined by measuring the level of the lysosomal enzymeHEXB secreted into the culture medium following treatment with drug. Asshown in FIG. 3, treatment with α-tocopherol, methyl-β-cyclodextrin, orcombinations of δ-tocopherol and methyl-β-cyclodextrin or α-tocopheroland methyl-β-cyclodextrin resulted in significant increases in lysosomalexocytosis as determined by HEXB secretions. The highest levels ofexocytosis were seen in Wolman fibroblasts treated with the combinationof δ-tocopherol and methyl-β-cyclodextrin.

To further characterize the effects of cyclodextrin on lysosome functionin fibroblast lines derived from patients with lysosomal storagediseases (Wolman, Tay-Sach, Farber, Battern, and Fabry), the effect ofcycoldextrin treatment on lysosomal Ca²⁺ release was determined. Bothwild-type and lysosomal storage disease fibroblasts were treated withcombinations of δ-tocopherol and methyl-β-cyclodextrin or α-tocopheroland methyl-β-cyclodextrin and the levels of lysosomal Ca²⁺ releasestimulated by 200 nM Gly-Phe β-naphthylamide (GPN) was measured. Asshown in FIG. 4, untreated lysosomal storage disease fibroblastsdisplayed reduced Ca²⁺ release compared to untreated wild-typefibroblasts. However, treatment of Wolman fibroblasts, Tay-Sachfibroblasts, Farber fibroblasts, Battern fibroblasts, and Fabryfibroblasts with combinations of δ-tocopherol and methyl-β-cyclodextrinor α-tocopherol and methyl-β-cyclodextrin restored lysosomal Ca²⁺release to those levels seen in wild-type fibroblasts.

The effect of various cyclodextrins, δ-tocopherol, and combinations ofcyclodextrins and δ-tocopherol on lipid accumulation and lysosome sizein wild-type and lysosomal storage disease fibroblasts (Wolman,Tay-Sach, Fabry, Farber, and MPSIIIB fibroblasts) was determined usingthe Lysotracker assay. In brief, cells were treated with variouscombinations and concentrations of drugs followed by staining with theLyso tracker dye which is a basophilic fluoresecent probe thataccumulates in acidic compartments, i.e. lysosomes, within the cell. Asshown in FIGS. 5A-5D, Lysotracker staining revealed enlarged lysosomesin Wolman, Tay-Sach, Fabry, Farber, and MPSIIIB fibroblasts compared towild-type cells. Treatment with δ-tocopherol or methyl-β-cyclodextrinalone reduced lipid accumulation and lysosome size whereas treatmentwith other cyclodextrins alone did not have a profound effect. Moreover,the combination of δ-tocopherol and methyl-β-cyclodextrin resulted in asignificant reduction in lipid accumulation and lysosome size.

Example 2 δ-Tocopherol and Cyclodextrin Treatment Resulted inAlleviation of the Pathological Ultrastructural Changes in Lysosomes ofCells with Lysosome Storage Disorders

The effects of δ-tocopherol and methyl-β-cyclodextrin on theultrastructural pathology of lysosomes in lysosome storage disordercells were investigated by electron microscopy. In brief, followingtreatment of wild-type and lysosome storage disorder fibroblasts withδ-tocopherol, methyl-β-cyclodextrin, or δ-tocopherol andmethyl-β-cyclodextrin, the fibroblasts were fixed and embedded. Thinsections of the embedded cells were then prepared and theultrastructural pathology was examined by electron microscopy. As shownin FIG. 6, untreated Wolman fibroblasts have lamellated and osmophilicstructures within the lysosomes. In addition, the cells also have thetypical elongated and cleft-shaped lipid droplets in the lysomes.However, these abnormal structures were significantly reduced bytreatment with δ-tocopherol and/or methyl-β-cyclodextrin. The effects ofδ-tocopherol and/or methyl-β-cyclodextrin on the ultrastructuralpathology of other lysosome disorder fibroblasts—Farber (FIGS. 7, 8, and10); Tay-Sachs (FIG. 8); Fabry (FIG. 8); Wolman (FIG. 9); NPA (FIG. 9);Batten (FIG. 9); MSIIIB (FIG. 9)—were analyzed by electron microscopy.As shown in FIGS. 7-10, treated cells appear as typical fibroblasts(elongated shape, well developed nuclei, normal mitochondria, swollensmooth and rough ER) but with significant amounts of endosomal (inparticular multivesicular bodies) and lysosomal compartments filled withlipid droplets and multilamellar bodies. These compartments are typicalfor Farber cells as seen before. Most cells have only small areas ofthese structures but a few cells are very much filled with thesestructures.

Example 3 Effect of Cyclodextrin in Single Use and in Combination withδ-Tocopherol in Human NPC1 Fibroblasts and Neural Stem Cells (NPC-NSCs)

In another set of experiments, in skin fibroblasts derived from NPC1patients, high concentrations of HBPCD (in millimolars) is needed forreduction of cholesterol accumulation and enlarged lysosomes (FIG. 11A).However, the small concentration of 50 uM of HBPCD in combination with10 uM delta-tocopherol reached the same effect as 5 mM HBPCD. Although160 uM MBCD almost completely reversed the phenotype of NPC1 cells, amuch smaller concentration of 20 uM MBCD in combination with 10 uMdelta-tocopherol reached the similar results as those obtained withhigher concentration of MBCD used along (FIG. 11B). Together, the dataindicate that MBCD is more potent (over 30 fold) than HBPCD forreduction of cholesterol accumulation and enlarged lysosome size in NPC1fibroblasts. In the combination with 10 uM delta-tocopherol, muchsmaller concentrations of HBPCD and MBCD are needed compared to thosewhen both drugs used along.

Example 4 Effect of Cyclodextrin in Single Use and in Combination withδ-Tocopherol in Neural Cells Derived from NPC1 Patients

Since major symptoms of NPC disease are within the central nervoussystem, the human NPC1 neuronal cells are better representative as a NPCdisease model for drug evaluation. Induced pluripotent stem cells(IPSCs) from the NPC1 skin fibroblasts were generated and differentiatedinto neural stem cells (NPC1-NSCs). In the Amplex-red cholesterol assay,the IC50 values of HBPCD and MBCD were 12 and 10 uM, respectively in theNPC1-NSCs, while the IC50 for delta-tocopherol was 18 uM (FIG. 12A). Inthe fluorescence microscopy experiments, the effects of HBPCD and MBCDon reduction of cholesterol accumulation (filipin staining) and enlargedlysosomes (Lysotracker staining) were also better than those ofdelta-tocopherol (FIG. 12B). Taken together, the data indicate thatHBPCD is much more potent in the human NPC1 neuronal cells than that inthe NPC1 skin fibroblasts, whereas the potency of delta-tocopherol isweaker in NPC1 neuronal cells compared to that in the NPC1 fibroblasts.

Example 5 Combination Therapy of Cyclodextrin and δ-TocopherolEffectively Reduced the Concentrations of Individual Compounds andIncreased the Effect on Reduction of Cholesterol Accumulation andEnlarged Lysosomes in NPC1-NSCs

A much reduced concentration of 50 uM HBPCD in combination with 10 uMdelta-tocopherol was determined to reach the same effect of 5 mM HBPCDused alone on reduction of cholesterol accumulation and enlargedlysosomes in the NPC1-NSCs (FIG. 13A). Similarly, the effect of 20 uMMBCD in combination with 10 uM delta-tocopherol was similar as 160 uMMBCD used alone in the NPC1-NSCs (FIG. 13B). The data demonstrate thatthe combination therapy of lower concentration of cyclodextrin anddelta-tocopherol could achieve the similar therapeutic effect onreduction of cholesterol accumulation and enlarged lysosomes in the NPC1neuronal cells as the large concentrations of both compounds when theyuse along. This concentration reduction of HBPCD or MBCD needed for thetreatment of NPC1 in combination with low concentration ofdelta-tocopherol may be important for the clinical use in patients.

Dosage recommendations taken from the studies using human NPC1 neutralstem cells (NPSCs) are as follows: (1) The IC50 value for HBPCD onreduction of cholesterol accumulation measured by the Amplex-redcholesterol assay is 50 uM and IC50 for delta-tocopherol is 15 uM. Thus,the ratio is 3.3 fold. (2) In combination therapy experiment, 50 uMHBPCD+10 uM delta-tocopherol significant reduced cholesterolaccumulation that is comparable with 5 mM HBPCD, while 50 uM HBPCD or 10uM delta-tocopherol along did not show the significant cholesterolreduction effect. (3) The molecule weight ratio of HBPCD (MW=1380.25)and delta-tocopherol (MW=402.65) is 3.4 fold.

Given a mouse body weight of 25 g, where brain:body ratio is 1:40, andassuming a complete distribution of HBPCD and delta-tocopherol afterdirect central envious system injection of compound byintracerebroventricular injection or intrathecal injection, a preferredsingle use of cyclodextrin for mammals including humans is from 0.1mg/Kg to 8 mg/Kg, more preferably 0.5 mg/kG or 1.0 mg/Kg to 2 mg/Kg, 3mg/Kg, 4 mg/Kg, 5 mg/Kg, 6 mg/Kg, 7 mg/Kg or 84 mg/Kg. One specificpreferred single use of cyclodextrin for mammals including humans is 3mg/Kg. In a combination therapy of a cyclodextrin compound administeredtogether or otherwise in conjuction with a vitamin E compound such asdelta-tocopherol, a preferred single dose for a mammal including a humanmay be from 0.05 to 1 mg/kg for each of the cyclodextrin compound andvitamin E compound (such as delta-tocopherol), more preferably 0.1 mg/Kgto 0.5 mg/Kg, 0.6 mg/Kg or 0.7 mg/Kg for each of a cyclodextrin compoundand vitam E compound such as delta-tocopherol, still more preferably 0.1mg/Kg to 0.3 mg/Kg or 0.4 mg/Kg for each of a cyclodextrin compound andvitam E compound such as delta-tocopherol.

Example 6 Effects of Cyclodextrin Single Use and Combination Therapy ofCyclodextrin with δ-Tocopherol

The effects of cyclodextrin in single use and in combination therapywith delta-tocopherol have been determined in patient derived skinfibroblasts with nine types of lysosomal storage diseases including NPC1(FIG. 14), Batten, Farber, ML III (FIG. 15), MLIV (FIG. 16), MPS1 (FIG.17), MPS VI (FIG. 18), NPA and Wolman disease. It was found that forsingle compound use, 8 mM HBPCD or 300 uM MBCD were needed for thesignificant effect on reduction of the enlarged lysosome size in thosecells. However, in a combination with 10 uM delta-tocopherol, 500 uMHBPCD or 20 uM MBCD significantly reduced enlarged lysosomes in thesecells. The results indicate an additive/synergistic effect ofcyclodextrin with delta-tocopherol on reduction of enlarged lysosomes inthe primary fibroblasts derived from patients with those nine lysosomalstorage diseases. The results also indicate that the dose ofcyclodextrin can be reduced 10 fold or more when it is used incombination with delta-tocopherol. The significant reduction ofcyclodextrin dose in combination with delta-tocopherol is important forthe treatment of lysosomal storage diseases because the high dose ofcyclodextrin may cause server side effects in prolonged treatmentprocess (it is possible that many of these patients may need a life timetreatment).

In addition, the high plasma and brain concentrations of cyclodextrinare difficult to achieve in the treatment of LSD patients. The 10 foldreductions of cyclodextrin concentration required in the combinationtherapy with delta-tocopherol makes the clinical use of cyclodextrin inLSD patients more feasible. Furthermore, delta-tocopherol is difficultto be dissolved in aqueous solution for use in patients that can beresolved in the combination therapy because cyclodextrin can facilitatedelta-tocopherol dissolving in solution.

Below is Table 1, showing the cell lines used for the above referencedstudies.

TABLE 1 Eponym Coriell of Disease Affected Accumulated Catalog diseasename Abbreviation gene Protein Lipid Genotype # Batten Ceroid CLN2 TPP1Tripeptidyl lipopigments p.R127X, GM16485 lipofuscinosis, peptidase I(lipofuscin) p.R208X neuronal 2 Fabry Alpha- GLA Alphaglobotriaosylceramide p.W162X, GM00107 galactosidase galactosidase Ars2071397 A deficiency rs2071228 Farber Lipogranulomatosis AC Acidceramidase ceramide p.Y36C, GM20015 (N-acylsphingosine p.Y36Camidohydrolase) Nieman NPC1 NPC1 NPC1 Unesterified p.P237S, GM03123n-Pick, cholesterol p.I1061T type C1 Nieman NPC2 NPC2 NPC2 Unesterifiedp.C93F, GM17910 n-Pick, cholesterol p.C93F type C2 Nieman NPA ASM AcidSphingomyelin p.L302P, GM16195 n-Pick, sphingomyelinase p.L302P type ASanfilippo Mucopolysaccharidosis MPS NAGL N-acetyl-alpha-D- Partiallydegraded p.R297X, GM02552 type III type B IIIB U glucosaminidase heparansulfate p.R643H B Tay- GM2 TSD Beta GM2 ganglioside c.1278ins GM00221Sachs gangliosidosis hexosaminidase TATC A c.1278ins TATC WolmanLysosomal LAL Lysosomal acid Cholesteryl ester unknown GM11851 acidlipase lipase & triglycerides deficiency

The contents of all references (including literature references, issuedpatents, published patent applications, and co-pending patentapplications) cited throughout this application are hereby expresslyincorporated herein in their entireties by reference. Unless otherwisedefined, all technical and scientific terms used herein are accorded themeaning commonly known to one with ordinary skill in the art.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents of the specificembodiments of the invention described herein. Such equivalents areintended with be encompassed by the following claims.

1. A method of treating a lysosomal storage disorder in a subject,comprising: determining that the subject is in need of non-cholesterollipid reduction or reduction of non-cholesterol dominant lipid andmacromolecule accumulation, and administering to the subject in needthereof; an effective amount of a cyclodextrin compound, or apharmaceutically acceptable salt, ester, solvate or hydrate thereof.2-4. (canceled)
 5. The method of claim 1, wherein the non-cholesterollipid is lipopigments, globotriaosylceramide, ceramide, sphingomyelin,heparan sulfate, partially degraded heparan sulfate, GM2 ganglioside,triglycerides, or cholesterol esters.
 6. The method of claim 1, whereinthe lysosomal storage disorder is Tay-Sachs disease, Sphingolipidoses,Gaucher disease, Mucolipidosis, Galactosialidosis, Salla disorder,Cystinosis, Danon disease, Fabry disease, Farber disease,Lipofuscinoses, Pompe disease, Gangliodisosis, ISSD, Krabbe disease,leukodystrophy, Hurler disease, Scheie disease, Hunter disease, SanFilippo disease, Sandhoff disease, Schinder disease, Batten disorder, orWolman disease.
 7. (canceled)
 8. The method of claim 1, wherein thecyclodextrin compound is of formula (I):

or a pharmaceutically acceptable salt, ester, solvate or hydratethereof, wherein, each R is independently H, alkyl, alkenyl, alkynyl,cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each of which isoptionally substituted; or —C(0)OR^(B), —OC(0)R^(B), C(0)R^(B), or—C(0)NR^(A)R^(B); each R₁ is independently H, alkyl, alkenyl, alkynyl,cycloalkyl, aryl, heteroaryl, halogen, hydroxy, amino, —CN, —CF₃, —N₃,—NO₂, —OR^(B), —SR^(B), —SOR^(B), —SO₂R^(B), —N(R^(B))S(0₂)—R^(B),—(R^(B))S(O₂)NR^(A)R^(B), —NR^(A)R^(B), —C(0)OR^(B), —0C(0)R^(B),—C(0)R^(B), —C(0)NR^(A)R^(B), or —N(R^(B))C(0)R^(B); each of which isoptionally substituted; each R^(A) is independently hydrogen, alkyl,alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl,each of which is optionally substituted; each R^(B) is independentlyhydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl,or heteroaryl, each of which is optionally substituted; n is 1, 2, 3, 4,5, 6, 7, 8, 9, or 10; and each m is independently 0, 1, 2, 3, 4, or 5.9. The method of claim 8, wherein each R is independently H, optionallysubstituted alkyl, —C(0)OR^(B), -0C(0)R^(B), —C(0)R^(B), or—C(0)NR^(A)R^(B).
 10. The method of claim 9, wherein each R isindependently H, methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, oroctyl; wherein each is straight chain or branched.
 11. The method ofclaim 8, wherein n is 1,2, or
 3. 12. The method of claim 1, wherein thecyclodextrin is 2-hydroxypropyl-β-cyclodextrin (2HPβCD),hydroxypropyl-β-cyclodextrin (HPβCD), methyl-β-cyclodextrin (MβCD),α-cyclodextrin, β-cyclodextrin, or γ-cyclodextrin, or a pharmaceuticallyacceptable salt, ester, solvate or hydrate thereof.
 13. The method ofclaim 1, further comprising the step of administering an additionaltherapeutic agent distinct from the cyclodextrin compound.
 14. Themethod of claim 13 wherein the additional therapeutic agent isadministered in combination or otherwise in conjunction with thecyclodextrin compound.
 15. The method of claim 13 wherein the additionaltherapeutic agent is a vitamin.
 16. The method of claim 15 wherein theadditional therapeutic agent is vitamin E. 17-18. (canceled)
 19. Themethod of claim 1 wherein the step of administering the cyclodextrincomprises administering the compound in a dosage of between about 0.01μg/kg and 100 mg/kg. 20-35. (canceled)
 36. A method of reducingnon-cholesterol lipids or reduction of non-cholesterol dominant lipidand macromolecule accumulation in a subject, the method comprisingadministering to the subject a cyclodextrin compound, or apharmaceutically acceptable salt, ester, solvate or hydrate thereof; anddetecting the amount of lipid reduction.
 37. The method of claim 36wherein the subject is identified as being in need of lipid reduction.38. The method of claim 1 further comprising the step of synthesizing orobtaining the cyclodextrin compound.
 39. The method of claim 1, whereinthe subject is a human.
 40. The method of claim 1 wherein thecyclodextrin compound and/or any additional distinct therapeutic agentsare administered intracranially to the subject.
 41. A pharmaceuticalcomposition comprising a cyclodextrin compound, or a pharmaceuticallyacceptable salt, ester, solvate or hydrate thereof, and vitamin E,together with a pharmaceutically-acceptable carrier or excipient. 42-44.(canceled)
 45. A kit comprising an effective amount of a cyclodextrincompound, or a pharmaceutically acceptable salt, ester, solvate orhydrate thereof, in unit dosage form, together with instructions foradministering the compound to a subject suffering from a lysosomalstorage disorder. 46-48. (canceled)